Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior.

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.     

Studies of individual carbon sites of hemoglobins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Proton-decoupled natural abundance 13C NMR spectra of carbon monoxide hemoglobins were recorded at 15.18 MHz by the Fourier transform method, under conditions of spectrometer sensitivity sufficient for detection of individual carbon resonances. The aromatic region of each spectrum contains broad bands of methine carbon resonances, and some relatively narrow peaks arising from nonprotonated carbons. Resonances of heme carbons were detected in spectra of carbon monoxide hemoglobins, but not in spectra of ferrihemoglobin (as a result of paramagnetic effects). Spectra of carbon monoxide hemoglobins from various species yielded only a few well resolved individual carbon resonances, most notably those of Cgamma of tryptophan residues. A comparison of the spectra of human adult, human fetal, chicken AII, and bovine fetal hemoglobins yielded specific assignments for all resonances of Cgamma of tryptophan residues. In the cases of human fetal, chicken AII, and bovine fetal hemoglobins, each tryptophan yielded a completely resolved individual carbon resonance. The chemical shift difference between the resonances of Cgamma of Trp-130beta and Cgamma of Trp-37beta is about 6 ppm. The chemical shift difference between Trp A12[14]alpha and Trp A12[15]beta is 1 ppm or less. A comparison of the chemical shifts of analogous tryptophan residues of the four carbon monoxide hemoglobins suggests very similar conformations in solution.     

Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy.

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of "solvent-exposed" tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cgamma of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 +/- 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. (i) Peak chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 coordinated to the metal. (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either. (iv) The gamma carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.     

Studies of individual carbon sites of hen egg white lysozyme by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Assignment of the nonprotonated aromatic carbon resonances to specific residues in the sequence.

The resonances of nonprotonated aromatic carbons in natural abundance 13C NMR spectra of hen egg white lysozyme are assigned to specific residues of the amino acid sequence. Chemical shift considerations, the effect of pH, and partially relaxed Fourier transform NMR spectra are used to assign each resonance to one of the seven types of nonprotonated aromatic carbons of amino acid residues. Spectra of chemically modified lysozyme samples yield various assignments to specific residues in the sequence. Line-broadening effects caused by binding of the relaxation probes Gd3+ and 4-N-acetamido-2,2,6,6-tetramethylipiperidine-1-oxyl yield specific assignments which are fully consistent with those based on chemical modifications. The effects of paramagnetic shift reagents and amino sugar inhibitors do not yield any obvious specific assignments. The effect of pH on the chemical shift of Cgamma of His-15 yields a pKalpha in agreement with published values, and indicates that the imidazole form of His-15 exists mainly (or entirely) as the Nepsilon3-H tautomer. The effect of pH on the chemical shifts (measured up to pH 8.8, at 38 degrees) of Czeta and Cgamma of the 3 tyrosine residues yields crude pKalpha values of 9.5 and 10 for Tyr-23 and one of the other tyrosines, respectively. The 3rd tyrosine residue does not exhibit titration behavior.     

Participation of tryptophan 62 in the self-association of hen egg white lysozyme. Application of natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Self-association of hen egg white lysozyme in solution of 38 degrees) is examined by means of natural abundance 13C nuclear magnetic resonance spectroscopy. The effect of pH on the resonances of the nonprotonated aromatic carbons of 9 mM lysozyme, and the effect of protein concentration (at pH 7) on these resonances, both indicate that self-association significantly affects the chemical shift of Cgamma of Trp-62, but not the chemical shifts of the other nonprotonated aromatic carbons. This result is consistent with the reported participation of Trp-62 in the intermolecular contact (Banerjee, S.K., Pogolotti, A., and Rupley, J.A. (1975) J. Biol. Chem. 250, 8260-8266). Our results indicate that the resonance of Cgamma or Trp-62 is a convenient monitor of lysozyme self-association. The chemical shift of this resonance reflects the extent of aggregation, while the line width yields information about the lifetime of the intermolecular contact. This lifetime is 1 to 2 ms at 38 degrees (9 mM protein, 0.1 M NaCl, pH 7). Our results also indicate that self-association of lysozyme is not accompanied by any general conformational change, and that binding of a lanthanide ion (at the metal ion binding site near the carboxylate groups of ASP-52 AND Glu-35) strongly suppresses self-association.     

Spinach plastocyanin: Comparison of reduced and oxidized forms by natural abundance carbon-13 nuclear magnetic resonance spectroscopy

Differences between the reduced Cu(I) and oxidized Cu(II) forms of spinach plastocyanin were investigated by natural abundance carbon-13 nuclear magnetic resonance spectroscopy at 67.9 MHz using proton noise decoupling. The spectra confirm that histidines 38 and 91 are copper ligands and demonstrate that coordination is by the N^o1 of both imidazole rings. Spectra of reduced plastocyanin yielded 128 separately resolved carbon resonances. Upon oxidation, 16 of these were observed to disappear; yet there was little change in the positions or intensities of other peaks. Those peaks which disappear are assigned to carbons near the metal. The protein evidently does not undergo a substantial change in conformation upon change of redox state.     

Proton resonance assignments and three-dimensional solution structure of the ragweed allergen Amb a V by nuclear magnetic resonance spectroscopy.

Essentially complete assignment of the proton resonances in the allergenic protein Amb a V has been made by analysis of two-dimensional NMR experiments. Conformational constraints were obtained in three forms: interproton distances derived from NOE cross-peak intensities of NOESY spectra, torsion angle constraints derived from J-coupling constants of COSY and PE-COSY spectra, and hydrogen bond constraints derived from hydrogen-exchange experiments. Conformations of Amb a V with low constraint violations were generated using dynamic simulated annealing in the program XPLOR. The refined structures are comprised of a C-terminal alpha-helix, a small segment of antiparallel beta-sheet, and several loops. A hydrophobic core exists at the interface of the alpha-helix and beta-sheet. The derived structure accounts for the several anomalous proton chemical shifts that are observed. The structure determined here for Amb a V is topologically similar to the structure determined previously for the homologous allergenic protein Amb t V [Metzler, W. J., Valentine, K., Roebber, M., Friedrichs, M. S., Marsh, D., & Mueller, L. (1992) Biochemistry 31, 5117-5127]; however, significant differences exist in the packing of side chains in the hydrophobic core of the molecules. Comparison of the detailed structural features of these two proteins will allow us to suggest surface substructures for the Amb V allergens that are likely to participate in B cell epitopes.     

Studies of chemically modified histidine residues of proteins by carbon 13 nuclear magnetic resonance spectroscopy. Reaction of hen egg white lysozyme with iodoacetate.

It is shown that natural abundance 13C NMR spectroscopy can be used to determine the structures and relative amounts of chemically modified forms of a histidine residue of a peptide or protein. The unfractionated product of the reaction of N alpha-acetyl-L-histidine with bromoacetate yields four resonances of nonprotonated aromatic carbons. These resonances are assigned (on a one-to-one basis) to C gamma of the intact amino acid, the two monocarboxymethylated derivatives (at N delta1 and N epsilon2), and the dicarboxymethylated derivative. The effect of pH on the chemical shift of C gamma is characteristic for each of the four species. This property is used to study the carboxymethylation of His-15 of hen egg white lysozyme upon treatment with iodoacetate. With the use of various reaction conditions, His 15 is carboxymethylated in detectable quantities only at N epsilon2. The spectra of the various reaction mixtures indicate which conditions are best for maximizing the yield of this derivative. A comparison of the spectrum of chromatographically pure [N epsilon2-carboxymethylhistidine-15]lysozyme with that of the intact protein indicates that the chemical modification does not significantly affect the conformation of the protein (at least in the regions of all aromatic amino acid residues).     

Application of nuclear magnetic resonance spectroscopy to proteins.

The active sites of enzymes can be studied in great detail using nuclear magnetic resonance spectroscopy. The determination of pKa values of active site histidine residues in bovine pancreatic ribonuclease and the characterization of the binding of peptide hormones to carrier proteins are two such examples. The study of the active site of staphylococcal nuclease is another example and is presented in detail in this paper. The structure of 3'5'-thymidine diphosphate bound in the active site of staphylococcal nuclease has been studied by measuring the relaxation rate enhancement of substrate analog nuclei by a paramagnetic metal ion. The lanthanide ion, Gd(III), was substituted for Ca(II) in the formation of the ternary complex of nuclease: Gd(III) : 3'5'-thymidine diphosphate. Measurements were made of the transverse relaxation rates of protons and the longitudinal and transverse relaxation rates of the phosphorus nuclei of bound nucleotide. Internuclear distances between the metal ion and atoms of the 3'5'-thymidine diphosphate nucleotide were determined from these data by using the Solomon-Bloembergen equation. In general, these distances corresponded closely to those determined by previous X-ray crystallography of the thymidine diphosphate complex. These internuclear distances were also used with a computer program and graphics display to solve for metal : nucleotide geometries which were consistent with the experimental data. A geometry similar to the structure of the metal : nucleotide complex bound to nuclease determined by X-ray analysis was one of the solutions to this computer modeling process. For staphylococcal nuclease the NMR and X-ray methods yield compatible high resolution information about the structure of the active site.     

Determination of three-dimensional structures of proteins and nucleic acids in solution by nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy has evolved over the last decade into a powerful method for determining three-dimensional structures of biological macromolecules in solution. Key advances have been the introduction of two-dimensional experiments, high-field superconducting magnets, and computational procedures for converting the NMR-derived interproton distances and torsion angles into three-dimensional structures. This article outlines the methodology employed, describes the major NMR experiments necessary for the spectral analysis of macromolecules, and discusses the computational approaches employed to date. The present state of the art is illustrated using a variety of examples, and future developments are indicated.     

Ribonuclease A: carbon-13 nuclear magnetic resonance assignments, binding sites, and conformational flexibility

Assignments have been made for 11 methyl, one Gln-C gamma, one Thr-C beta, and all six Tyr-C zeta carbon resonances of ribonuclease A. These partially serve to delineate the binding sites for Cu2+, Mn2+, phosphate, cytidine and its 2'-, 3'-, and 5'-phosphates (Cyd and Cyd-2'-P, -3'-P, and -5'-P), and one or a few urea molecules at low concentration. Evidence is presented for a conformational change, and hence flexibility, in the active site region around the optimum pD for enzymic activity and another such change at around the optimum temperature. The binding of cytidine-containing ligands is shown to have extensive conformational consequences for methyl groups but less for hydrophobic aromatic residues, implying that the former make a special contribution to molecular flexibility. The cytosine ring in Cyd-2'-P, -3'-P, and -5'-P is found to be close but far from parallel to the ring of Phe-120. In contrast to previous claims, ribonuclease A is shown not to unfold even partially before denaturation. On denaturation, it passes to a new but structured state.